1. Field of the Invention
The present invention relates to a method for the preparation of maltooligosaccharide glycoside derivatives of p-nitrophenol, 4-methylumbelliferone and other chromogenic or fluorogenic aglycones. More particularly, the invention relates to an enzymatic method for the preparation of suitable chromogenic or fluorogenic maltooligosaccharide glycoside derivatives for use, for example, as substrates for the determination of amylase activity.
2. Description of the Prior Art
Heretofore, only processes involving organic synthetic reactions have been used for the preparation for the chromogenic or fluorogenic derivatives of oligosaccharides suitable for use as substrates for the determination of amylase activity. (Jansen and Rawlins, Nature 182, 525, 1958; and Driscoll, Richard C., et al., U.S. Pat. No. 4,102,747. In these methods, the yield of desired derivative, its suitability as an amylase substrate, or its low purity have prevented significant improvements in several analytical systems for the determination of amylase activity which would be possible if such substrates were available in a commercially and technically acceptable fashion.
Amylase assay procedures have been developed which utilize substrates such as starch, glycogen, dextrin, and oligosaccharides. However, the use of such substrates has suffered from the disadvantage that endogenous glucose present in the reaction sample sometimes causes interference with the results of the assay. Procedures utilizing these substrates have been developed which overcome the glucose interference problem, however the reaction sequences involved in these procedures are complicated and often quite costly.
The Driscoll, et al. procedure cited above overcomes the problems of glucose interference and high cost, however, heretofore no satisfactory procedure has been reported for producing the maltooligosaccharide glycoside substrate in a commercially feasible manner. The availability of defined chromogenic or fluorogenic derivatives of maltooligosaccharide glycosides will permit significant improvements in the Driscoll et al. method for the determination of amylase activity according to the following reactions in which the substrate, p-nitrophenyl-.alpha.-D-maltoheptaoside is used for illustrative purposes only: ##STR1##
The rate of formation of p-nitrophenol, once zero-order kinetics is established for equation (2) is directly proportional to the amylase present in the sample. p-Nitrophenol may be monitored spectropholometrically by its absorbance at 405 nm.
In a similar fashion, amylase activity may be determined using a fluorogenic substrate such as 4-methylumbelliferyl-.alpha.-D-maltoheptaoside. In such a case, the rate of increase in fluorescence is proportional to the amylase activity.
The organic synthetic reactions heretofore used for producing chromogenic and fluorogenic substrates have produced substrates having both .alpha. and .beta. configurations. Accordingly, because in the Driscoll et al. method the use of substrates having the .beta. configuration requires the use of .beta. glucosidase as an additional reactant, there is a need for a method for producing substrates having either the .alpha. configuration or the .beta. configuration in substantially pure form. The availability of substrates with the .beta.-anomeric configuration would provide improvements in the Driscoll et al. method for the measurement of amylase activity according to the following reactions in which the substrate, p-nitrophenol-.beta.-D-maltoheptaoside is used for illustrative purposes only: ##STR2## The rate of formation of p-nitrophenol, once zero-order kinetics has been established, for reaction (3) is directly proportional to the amylase activity of the sample. p-Nitrophenol may be monitored by its absorbance at 405 nm spectrophotometrically. In a similar fashion, amylase activity can be determined using a fluorogenic substrate such as 4-methylumbelliferyl-.beta.-D-maltoheptaoside. In such a case, the rate of increase in the fluorescence is proportional to the amylase activity.
From the foregong discussion, it can be readily seen that the analytical methods employing chromogenic or fluorogenic substrates for the measurements of amylase activity represent decided improvements in existing technology for the measurment of the enzyme. To render these procedures useful for routine use, there is a need for methods for producing the .alpha. and .beta.-maltooligosaccharide glycoside derivatives in substantially pure form and high yield.
The well known glucanotransferase (E.C.2.4.1.19) of Bacillus macerans has primarily been used for the production of cyclic dextrins from starch. (Tilden and Hudson; J. Bact., 43, 727-744, 1942, J. Am. Chem. Soc., 61, 2900-2902, 1939). The use of the enzyme for the transfer of glucosyl groups from cyclic dextrins to suitable acceptors such as D-glucose, D-maltooligosaccharides, and D-glucosides, has been reported (French, et al; J. Am. Chem. Soc. 76 2387-2390, 1954). Also, in British Pat. No. 1,442,480, is reported a method for producing oligoglucoylfructose utilizing a glucanotransferase enzyme isolated from a strain of Bacillus stearothermophilus. A similar enzyme has been isolated from Klebsiella pneumoniae (Bender, Arch. Microbiol., 111, 271-282, 1977) and from Bacillus megaterium (Kitahata, et al., Proc. Symposium on Amylase, Volume 7, 61-68, 1972). Heretofore, the use of glucanotransferase enzymes for the enzymatic synthesis of substantially pure .alpha. and .beta. p-nitrophenyl or 4-methylumbelliferyl maltooligosaccharide glycoside derivatives has not been reported.